Discoveries in the basic realm of molecular biology over the past ten years have led to the realization that RNA has a series of distinct capabilities and biological activities previously unsuspected. The most important of these novel RNA-level discoveries has been the finding that RNA can be an enzyme as well as an information carrier.
Various RNA molecules have one or more enzymatic activities, such as an endoribonuclease activity which acts to cleave other RNA molecules. Such activity is termed intermolecular cleaving activity. These enzymatic RNA molecules are derived from an RNA molecule which has an activity which results in its own cleavage and splicing. Such self-cleavage is an example of an intramolecular cleaving activity.
Since 1982, several unexpected diseases caused by RNA-based pathogenic agents have emerged. These include the lethal Acquired Immune Deficiency Syndrome (AIDS) and delta hepatitis (also called Hepatitis D), a particularly virulent form of fulminant hepatitis caused by a viroid-like RNA agent. These blood-borne diseases are spread at the RNA level, manifest themselves in cells of patients, and are by now present within the bloodstream of millions of individuals.
Conventional biotechnology, with its reliance on recombinant DNA methods and DNA-level intervention schemes, has been slow to provide valid approaches to combat these diseases.
The potential of ribozymes (RNA enzymes) to catalyze the cleavage of RNA substrates makes them attractive molecular tools. Ribozymes are an interesting alternative to RNA interference approach that seems to trigger immunological responses. Many efforts were directed at increasing the substrate specificity of ribozyme cleavage, which can be considered as a limit to their utilization. For example, allosteric ribozymes for which the catalytic activity is regulated by an independent effector, have been developed.
Delta ribozymes, derived from the genome of hepatitis delta virus (HDV), are metalloenzymes. Like other catalytically active ribozymes, namely hammerhead and hairpin ribozymes, the delta ribozymes cleave a phosphodiester bond of their RNA substrates and give rise to reaction products containing a 5′-hydroxyl and a 2′,3′-cyclic phosphate termini. Two forms of delta ribozymes, namely genomic and antigenomic, were derived and referred to by the polarity of HDV genome from which the ribozyme was generated. Both HDV strands forms exhibit self-cleavage activity, and it has been suggested that they are involved in the process of viral replication. This type of activity is described as cis-acting delta ribozymes.
Like other ribozymes, delta ribozymes have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting either a specific mRNA or viral RNA molecule. A very low concentration (<0.1 mM) of Ca2+ and Mg2+ is required for delta ribozyme cleavage.
With respect to the structure of the δ ribozyme, it folds into a compact secondary structure that includes pseudoknots (for reviews see Bergeron et al., Current Med. Chem. 10, 2589-2597, 2003). This structure is composed of one stem (the P1 stem), one pseudoknot (the P2 stem is a pseudoknot in the cis-acting version), two stem-loops (P3-L3 and P4-L4) and three single-stranded junctions (J1/2, J1/4 and J4/2). Both the J1/4 junction and the L3 loop are single-stranded in the initial stages of folding, but are subsequently involved in the formation of a second pseudoknot that consists of two Watson-Crick base pairs (the P1.1 stem). In terms of general organization, the P1 and P3 stems, along with the J4/2 junction, form the catalytic center, while the P2 and P4 stems are located on either side of the catalytic centre and stabilize the overall structure.
The binding domain of δRz (the P1 stem) is composed of one G-U wobble base pair followed by six Watson-Crick base pairs. In addition, the nucleotides from position −1 to −4 of the substrate, that is those adjacent to the scissile phosphate, were shown to contribute to the ability of a substrate to be cleaved efficiently. Thus, the substrate specificity of δRz cleavage is based on a total of 11 nucleotides, which might be a limiting factor when trying to specifically target an RNA species in a cell. Because the P1 stem is located within its catalytic center, all attempts to modify the length of this stem result in the loss of catalytic ability.
In International publication WO99/55856 (Jean-Pierre Perreault et al.), the entire content of which is hereby incorporated by reference, filed in the name of Université de Sherbrooke, there is disclosed a nucleic acid enzyme for RNA cleavage, and more particularly a delta ribozyme and mutants thereof.
In U.S. Pat. No. 5,225,337 (Hugh D. Robertson et al.), issued on Jul. 6, 1993, there are disclosed ribozymes derived from a specific domain present in the hepatitis delta virus (HDV) RNA for specifically cleaving targeted RNA sequences and uses thereof for the treatment of disease conditions which involve RNA expression, such as AIDS. These ribozymes consist in at least 18 consecutive nucleotides from the conserved region of the hepatitis delta virus between residues 611 and 771 on the genomic strand and between residues 845 and 980 on the complementary anti-genomic strand. These ribozymes are proposed to fold into an axe-head model secondary structure. According to this model structure, these ribozymes require substrate base paired by 12-15 nucleotides. More specifically, a substrate bound to the ribozyme through the formation of two helices. A helix is located upstream to the cleavage site (i.e. in 5′ position) while the second helix is located downstream to the cleavage site (i.e. in 3′ position).
In U.S. Pat. No. 5,625,047 (Michael D. Been et al.), issued on Apr. 29, 1997, there are disclosed enzymatic RNA molecules proposed to fold into a pseudoknot model secondary structure. These ribozymes were proposed to cleave at almost any 7 or 8 nucleotide site having only a preference for a guanosine base immediately 3′ to the cleavage site, a preference for U, C or A immediately 5′ to the cleavage site, and the availability of a 2′ hydroxyl group for cleavage to occur. The specificity of recognition of these ribozymes is limited to 6 or 7 base pairing nucleotides with the substrate and a preference of the first nucleotide located 5′ to the cleavage site. Neither tertiary interaction(s) between the base paired nucleotides and another region of the ribozyme, nor single-stranded nucleotides are involved to define the specificity of recognition of these ribozymes. Because the recognition features were included in a very small domain (i.e. 6 or 7 base paired nucleotides) in order to exhibit the desired activity, these ribozymes have a limited specificity, and thus, not practical for further clinical applications.
It would be highly desirable to be provided with a new ribonucleic acid, target-dependent adapter to increase specificity of the nucleic acid for its target.